S-Layer Anchoring and Localization of an S-Layer-Associated Protease in Caulobacter crescentus

Bacterial strains, plasmids, and growth conditions.All of the strains and plasmids used in this study are listed in Tables 1 and 2, respectively. E. coli DH5α was used for most E. coli cloning manipulations and was grown at 37°C in Luria broth (1% tryptone, 0.5% NaCl, 0.5% yeast extract) with 1.3% agar for plates. C. crescentus strains were grown at 30°C in PYE (0.2% peptone, 0.1% yeast extract, 0.01% CaCl₂, 0.02% MgSO₄) medium with 1.2% agar for plates. Ampicillin, kanamycin (KAN), and streptomycin were used at 50 μg/ml, and chloramphenicol (CHL) was used at 20 μg/ml for E. coli and 2 μg/ml for C. crescentus. KAN and streptomycin were used at 25 μg/ml when used along with CHL for C. crescentus.

Plasmid and DNA manipulations.Standard methods of DNA manipulation were used (29). Electroporation of C. crescentus was performed as previously described (20). PCR products were generated using Platinum Pfx DNA polymerase (Invitrogen). Sequences of the primers used in this study are detailed in Table 3. Plasmids to be digested with ClaI were produced from RB404, a non-DNA methylating strain of E. coli.

C. crescentus expression vectors. (i) p4A.p4A is a vector derived from pUC8 (25) that contains a full-length copy of rsaA under control of a modified rsaA promoter region. It contains an oriV-derived plasmid RSF1010 (39), which allows for replication in E. coli and in C. crescentus strains that express repBAC. Oligonucleotides 1060 and 1920 were used for inverse PCR of pUC8. The PCR product was digested with StuI and ScaI and ligated to the HpaI-digested Cm resistance gene called CHE, resulting in pUC8CX. CHE was made using the oligonucleotides JNCHE-1 and JNCHE-2 to PCR amplify the gene from pMMB206 (26). The oriV was inserted by digesting pUC8CX with EcoO109, filling in the resulting recessed ends with Klenow polymerase, and then digesting with HindIII; this small fragment was replaced with the 521-bp HindIII/XmnI oriV fragment from pCR2.1oriV (39), resulting in pUC8CVX. The lac promoter was replaced with a modified rsaA promoter region as follows: the EcoRI/HindIII fragment from pSSa49ΔSD (10) containing a modified rsaA promoter region was ligated into pUC8, resulting in pUC8ΔSD. pUC8ΔSD and pUC8CVX were digested with EcoRI and ligated. This plasmid was digested with SapI and PstI, filled in with Klenow polymerase, ligated, and selected on CHL. This last manipulation removed all of pUC8ΔSD except the modified rsaA region, now upstream of the EcoRI/HindIII multiple cloning site region. This plasmid, pUC8CVXΔSD, was renamed p4A for simplicity. All full-length rsaA constructs can be cloned into this high-copy-number vector as EcoRI/HindIII fragments and transcribed from the modified rsaA promoter region. Wild-type (WT) rsaA from pTZ18UB:rsaAΔP (8) in p4A is p4A-WT.

(ii) pBBR3ΔSD. pBBR3ΔSD was used as a E. coli and C. crescentus shuttle vector and does not require repBAC to replicate. To create this vector, p4A-WT was digested with NspI, and the recessed ends were filled in with Klenow polymerase. The linearized plasmid was ligated into pBBR3 (36) digested with SmaI, creating a 12-kb intermediate plasmid. The plasmid was digested with HindIII, releasing most of p4A-WT, leaving the modified rsaA promoter region with pBBR3 vector. The remaining fragment was circularized by ligation, resulting in pBBR3ΔSD.

Construction of plasmids carrying rsaA with collagenase cleavage sites. (i) pUC9CXSMCC.pUC9CXSMCC was created by annealing oligonucleotides MCC-1 and MCC-2 and ligating them into XhoI-StuI-digested pUC9CXS (7) in the same orientation as the promotorless Cm resistance gene. These oligonucleotides encode, from 5′ to 3′, a factor X protease cleavage site, followed by two collagenase cleavage sites in tandem (PHGPAGP).

(ii) pWB9:rsaAΔP:Hps1MCCΔ, pWB9:rsaAΔP:Hps4MCCΔ, and pWB9:rsaAΔP:Hps12MCCΔ.pUC9CXSMCC was digested with BamHI, releasing a fragment that included the MCC and a promotorless Cm resistance gene. The BamHI cassette was then ligated into the unique BamHI site in either pTZ18UB:rsaA(HinPI277BamHI) (7), pTZ18UB:rsaA(HinPI690BamHI) (7), or pTZ18UB:rsaA(HinPI723BamHI) (7) to place the MCC cassette at the position in rsaA corresponding to amino acid 277, 690, or 723, respectively. Proper orientation of the MCC cassette was achieved by selecting for Cm resistance (the Cm resistance gene is driven by the lac promoter in pTZ18). The Cm resistance gene was then excised by digestion with BglII, and plasmids were recircularized by ligation, creating intermediate plasmids. The EcoRI-SstI fragments of these plasmids were excised and ligated into pWB9, resulting in pWB9:rsaAΔP:Hps1MCCΔ, pWB9:rsaAΔP:Hps4MCCΔ, and pWB9:rsaAΔP:Hps12MCCΔ.

(iii) pWB9:rsaAΔP:B162MCCΔ.pBSKIIφBamHI was constructed by cutting pBSKII with BamHI and filling in with Klenow polymerase, and the product was religated, thereby destroying the BamHI site. Next, the rsaA EcoRI/ClaI fragment was excised from pTZ18UB:rsaA(HinPI723BamHI) (7) and ligated into pBSKIIφBamHI. Mutation at RsaA amino acids 162/163 was achieved using Quickchange (Stratagene, La Jolla, CA), using the primers B162F and B162R, changing RsaA amino acids F162/L163 to G162/S163 and creating a BamHI site. The MCC cassette from pUC9CXSMCC was inserted into this BamHI site and the Cm resistance gene was removed as described above, yielding pBSKIIφBamHI:rsaAEcoRI-ClaIBamHI162MCCΔ. p4A-WT was digested with PstI, releasing a PstI-PstI fragment containing two ClaI sites from a noncoding region of the plasmid. The remaining backbone plasmid was recircularized by ligation, creating p4A-WTΔPstI. Next, the rsaA EcoRI/ClaI fragment with the desired mutation, excised from pBSKIIφBamHI:rsaAEcoRI-ClaIBamHI162MCCΔ, was inserted by ligation into p4A-WTΔPstI. To replace the PstI-PstI fragment in this intermediate plasmid (which contained an SstI site required for the next step), the AvrII/HindIII fragment (containing the PstI-PstI fragment), obtained from digesting p4A-WT with AvrII and HindIII, was ligated into the intermediate plasmid. Finally, the mutant rsaA-encoding EcoRI/SstI fragment was ligated into pWB9 as described above, yielding pWB9:rsaAΔP:B162MCCΔ.

Construction of plasmids carrying rsaA with collagenase cleavage at RsaA position 277 with additional mutations in the RsaA N terminus. (i) pWB9:rsaAΔP:B7Hps1MCCΔ, pWB9:rsaAΔP:B154Hps1MCCΔ, and pWB9:rsaAΔP:B222Hps1MCCΔ.pTZ18UB:rsaA(HinPI277BamHI)MCCΔ was used as a template for PCR for site-directed mutagenesis of rsaA to achieve “BamHI” mutations at RsaA positions 7, 154, and 222 using Quickchange (Stratagene). Primers B7F and B7R were used to change RsaA Q7/L8 to G7/S8, primers B154F and B154R were used to change V154/D155 to G154/S155, and primers B222F and B222R were used to change D222/L223 to G222/S223. The EcoRI-SstI fragments from the Quikchange products were excised and ligated into pWB9 as described above, yielding pWB9:rsaAΔP:B7Hps1MCCΔ, pWB9:rsaAΔP:B154Hps1MCCΔ, and pWB9:rsaAΔP:B222Hps1MCCΔ.

(ii) pWB9:rsaAΔP:Taq29Hps1MCCΔ, pWB9:rsaAΔP:Mps4Hps1MCCΔ.The EcoRI-NotI fragments from pTZ18UB:rsaA(TaqI29BamHI) (8) or pTZ18UB:rsaA(MspI69BamHI) (7) were ligated into pTZ18UB:rsaA(HinPI277BamHI)MCC. The rsaA EcoRI/SstI fragments were cloned into pWB9 as described above, yielding pWB9:rsaAΔP:Taq29Hps1MCCΔ and pWB9:rsaAΔP:Mps4Hps1MCCΔ.

(iii) pBBR3ΔSD:Taq169Hps1MCCΔ.pTZ18UB:rsaA (HinPI277BamHI)MCCΔΔPstI was constructed as described above to remove two ClaI sites in the noncoding PstI-PstI fragment. The rsaA EcoRI/ClaI fragment from pTZ18UB:rsaA(TaqI169BamHI) (8) was ligated into pTZ18UB:rsaA (HinPI277BamHI)MCCΔΔPstI. From the resulting plasmid, the rsaA EcoRI/HindIII fragment was ligated into pBBR3ΔSD, yielding pBBR3ΔSD:Taq169Hps1MCCΔ.

Plasmids used for gene disruptions. (i) pK18mobsacBmanBΔNΔC.pK18mobsacBmanBΔNΔC was used to create a strain that was null for ManB and, consequently, SLPS. A PCR product encoding manB that was deleted in the regions encoding the N and C termini of ManB was generated using NA1000 chromosomal DNA and the primers ManB 169 and IManB 1202 and then blunt-end ligated into EcoRV-cut pBSKIIEEH (37). The EcoRI/HindIII fragment from the resulting plasmid was then ligated into pK18mobsacB (31), yielding pK18mobsacBmanBΔNΔC.

(ii) pK18mobsacB:pwbΔR.pK18mobsacB:pwbΔR was used to create an internal deletion in the S-layer-associated protease gene, sap. A PCR product encoding sap was created using JS4000 chromosomal DNA as the template and primers PWB_F and PWB_R and blunt-end ligated into EcoRV-digested pBSKII. sap was excised from the resulting plasmid as an EcoRI/HindIII fragment and ligated into pBSKIIESH (37). The resulting plasmid was digested with PstI, which removed 1,023 bp of the sap sequence (approximately one-half of the sap gene), and the remaining plasmid was recircularized by ligation. The internally deleted form of sap was excised as an EcoRI/HindIII fragment and ligated into pK18mobsacB, yielding pK18mobsacB:pwbΔR.

(iii) pK18mobsacB:rsaF _aΔKP.pK18mobsacB:rsaF _aΔKP was used for the internal deletion of rsaF _a. A PCR product containing rsaF _a and flanking regions of 1,008 bp 5′ and 139 bp 3′ was generated using NA1000 chromosomal DNA as a template and primers rsaF _aΔKP_F and rsaF _aΔKP_R. This fragment was blunt-end ligated into the pBSKI+ plasmid at the EcoRV site, resulting in pBSKI:rsaF _aEX. pBSKI:rsaF _aEX was digested with KpnI and PstI, blunt ended using T4 DNA polymerase, and recircularized by ligation. The resulting plasmid had an 852-bp internal deletion. This deleted version of rsaF _a was excised as an EcoRI-HindIII fragment and ligated into pK18mobsacB, yielding pK18mobsacB:rsaF _aΔKP.

(iv) pK18mobsacBrsaA353ΦB.pK18mobsacBrsaA353ΦB was used to disrupt the rsaA gene. The BamHI site in pTZ18UB:rsaA(AciI353BamHI) (7) was destroyed by digesting with BamHI and filling in with Klenow polymerase, and then the product was recircularized by ligation. This rendered the rsaA sequence out of frame and introduced an amber codon at a position corresponding to RsaA amino acid 358. The EcoRI/HindIII fragment containing the mutated rsaA gene was then ligated into pK18mobsacB, yielding pK18mobsacBrsaA353ΦB.

Construction of gene disruptions. (i) JS1010.Knockout of rsaF _a in NA1000 was done using pK18mobsacB:rsaF _aΔKP. Primary recombination of the plasmid was selected using Km resistance. Secondary selection on 5% sucrose PYE plates and subsequent replica plating on PYE and PYE KAN plates were used to confirm a second recombination event. rsaF _a deletion was confirmed by PCR using primers JS1010_F and JS1010_R.

(ii) JS1011.Knockout of rsaF _a in JS1008 (36) was done as described above for the knockout of rsaF _a in NA1000, except that colonies were screened by colony Western blot analysis (7) using RsaA antiserum. Colonies that were S-layer negative according to the colony Western blot analysis were confirmed by PCR. A strain possessing the internally deleted rsaF _a and CHL cassette-interrupted rsaF _b was then subjected to rsaA deletion, using pAL1. The deletion was confirmed by PCR using primers JS1011_F and JS1011_R.

(iii) JS1012.pK18mobsacB:pwbΔR was used to knockout the sap gene in JS1001 (15), replacing the wild-type gene with an internally deleted version of sap missing about one-half of the gene sequence. Recombinants were selected as described for JS1010. Gene replacement was confirmed by PCR using primers PWB_F and PWB_R. The rsaA gene was deleted using pAL1. Recombinants were selected as described for JS1011. Colonies were screened by PCR using primers JS1011_F and JS1011_R.

(iv) JS1013.pK18mobsacBrsaA353ΦB was used to disrupt rsaA in NA1000. Recombinants were selected for as described for JS1010 and were screened by colony Western blot analysis (7) using RsaA antiserum. Loss of RsaA was confirmed by low-pH extraction (see below).

(v) JS1014.pK18mobsacBmanBΔNΔC was used to knock out manB in JS1013. Recombination of pK18mobsacBmanBΔNΔC was selected for using Km resistance.

(vi) JS4022.JS4022 was constructed by introduction of repBAC into recA of JS4015 in the same way that resulted in JS4019, as described previously (39).

(vii) JS4023.Knockout of rsaF _a in JS4000 was done using pK18mobsacB:rsaF _aΔKP as described for creation of JS1010.

(viii) JS4024.Knockout of manB in JS4015 (38) was achieved using pK18mobsacBmanBΔNΔC in the same manner used to create strain JS1014.

(ix) JS4025.Knockout of rsaF _b in JS4023 was done via insertional inactivation using an N- and C-terminally deleted rsaF _b fragment. The nonreplicating pTZ18UCHE:rsaF _bΔNΔC (37) was electroporated into JS4023, selecting for Cm resistance. rsaF _b insertion was confirmed by PCR using primers 1060 and JS4025_R.

S-layer reattachment assays. (i) Coculturing assay.Coculturing of S-layer donors (JS1001) with S-layer recipients (JS1003 and JS1004) was done as follows. Strains JS1001, JS1003, and JS1004 were grown to mid-log phase. An aliquot of JS1001 and either JS1003 or JS1004 in a cellular ratio of eight donors:one recipient was used to inoculate 10 ml of PYE medium. These cocultures were grown to mid-log phase and filtered through Whatman no. 52 hardened filter paper (Kent, England) to remove any aggregated protein. Equivalent numbers of cells (assessed by the optical density at 600 nm [OD₆₀₀]) were then subjected to low-pH extraction (see “Protein techniques” below) to assess the extent of S-layer reattachment.

(ii) RsaA reattachment assay.To produce soluble protein for reattachment purposes, 10-ml cultures of SLPS-negative cultures (JS1001, for wild-type RsaA) or of SLPS-negative, S-layer-negative C. crescentus strains that are Sap positive (JS1004) or Sap negative (JS1012) harboring the plasmids encoding RsaA or RsaA mutants were grown to an OD₆₀₀ of ∼0.9 and pelleted by centrifugation three times at 13,000 ×g for 10 min at 4°C; the pellet was discarded after each centrifugation in an effort to clear the supernatant of cells. S-layer-negative target cells possessing various levels of SLPS (JS1003, JS1004, and JS4024) were grown to an OD₆₀₀ of ∼0.9 and then pelleted by centrifugation at 13,000 × g for 5 min at 4°C. Cell pellets were washed with 1 ml of PYE medium, subjected to centrifugation, and then suspended with the supernatants containing the soluble RsaA or RsaA mutant. The volume of supernatant used for resuspension was 1.2 to 1.5 times the volume of the target cells initially pelleted. The resulting mixtures were incubated at room temperature for 10 to 30 min with slow inversion. These cultures were then pelleted, washed twice with 10 mM HEPES (pH 7) for subsequent low-pH extraction or washed twice with 10 mM Tris-HCl (pH 8) for subsequent boiling of the entire sample or for subsequent whole-cell protein preparations.

Protein techniques. (i) Low-pH extraction.Surface protein from C. crescentus cells was extracted by low-pH extraction or by EGTA treatment as previously described (41). To compare the amounts of S-layer protein from C. crescentus cells expressing RsaA to S-layer that had been reattached to S-layer-negative cells, equivalent amounts of cells (determined by the OD₆₀₀) growing at log phase were used, and equal amounts of extracted protein samples were loaded onto protein gels.

(ii) Whole-cell protein preparations.Whole-cell protein preparations were done using equivalent amounts of cells (determined by the OD₆₀₀) taken from 10-ml C. crescentus cultures growing at log phase. Cultures were centrifuged, and the cell pellets were washed twice with 10 mM Tris-HCl, pH 8. The cells were resuspended in 100 μl of 10 mM Tris-HCl, pH 8, and lysozyme (100 μg/ml) was added; the mixture was then incubated at 25°C for 15 min. RNase A (50 μg/ml) and DNase I (1 μg/ml) were added, and incubation continued at 37°C for 30 min. Equal volumes of whole-cell protein preparations were loaded onto protein gels. Some samples were subjected to low-pH extraction before whole-cell protein preparation was done to disrupt RsaA monomer-monomer interactions in order to assess RsaA that was directly anchored to the cell surface. As a rapid alternative to this method, for some experiments equivalent numbers of cells were washed twice with 10 mM Tris-HCl, pH 8, and boiled for 5 min.

(iii) Collagenase digests.Collagenase digests performed on soluble RsaA carrying a collagenase recognition sequence were done in PYE medium supplemented with collagenase buffer as follows. Supernatant from S-layer-shedding C. crescentus strains were harvested as described above (see “RsaA reattachment assay”). The resulting culture supernatant was supplemented to achieve final concentrations of 10 mM NaCl, 4 mM CaCl₂, 2 mM Tris-HCl (pH 7), and 2 mM β-mercaptoethanol. Type III collagenase (Sigma-Aldrich, St. Louis, MO) was added to 7 U/ml, and digestion was performed for various lengths of time at 37°C.

(iv) SDS-PAGE and Western blotting.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) used 7.5%, 10%, or 12% separating gels as appropriate for the anticipated protein sizes. Coomassie-stained SDS-PAGE gels and Western immunoblotting were done by standard methods (29); for Western analysis horseradish peroxidase-coupled secondary antibodies were used for colorimetric detection. Antiserum of RsaA with a deletion of residues 188 to 784(37) was used at a 1/10,000 dilution, SLPS antiserum (40) was used at a 1/6,000 dilution, and RsaF_a antiserum (22), which was raised against RsaF_a but is also reactive with RsaF_b, was used at a 1/1,000.

Comparison of RsaA preparation methods for cell surface reassembly.One goal in assay development was to find a method of RsaA preparation that allowed for optimum levels of RsaA reassembly onto S-layer-negative cells. Full-length RsaA can be obtained by treating cells to low-pH conditions or EGTA extraction (41). Alternatively, SLPS-deficient strains shed RsaA into the culture medium, and much of this protein forms aggregates (2) that can be collected by filtration and solubilized by urea treatment. Previously established methods of RsaA preparation for subsequent reassembly onto lipid vesicles involved low pH and EGTA treatment of cells to release RsaA (27). Therefore, low-pH- and EGTA-extracted RsaA preparations were incubated with S-layer-negative cells that possessed either wild-type (JS1003) or deficient (JS1004) levels of SLPS in the normal C. crescentus growth medium PYE. RsaA obtained from both extraction methods reattached to cells in an SLPS-dependent manner (except for the EGTA-extracted RsaA that was not dialyzed against water prior to addition to S-layer-negative cells, probably due to a shortage of available Ca²⁺) (Fig. 1a). Slightly more low-pH-extracted protein appeared to reassemble in comparison to EGTA-extracted protein; however, neither approach led to S-layer levels comparable to those found on wild-type cells (Fig. 1a). Similar results were obtained when cells and protein were incubated in calcium-supplemented water rather than PYE medium (not shown).

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FIG. 1.

Anti-RsaA immunoblot analysis of S-layer reattachment onto C. crescentus cells. (a) S-layer reattachment. Wild-type RsaA was extracted from the C. crescentus cell surface by low-pH or EGTA treatment, and the resulting protein was dialyzed (dial) or not against water. These RsaA preparations were subsequently incubated with corresponding amounts of S-layer-negative cells that possess wild-type (JS1003; wt) or deficient (JS1004; d) levels of SLPS. Reattached RsaA was subsequently extracted by low-pH treatment. For comparison, the lane labeled as control (ctl) is a low-pH extraction of an equivalent number of cells expressing wild-type levels of S-layer. N/A, not applicable. (b) S-layer reattachment by coculturing S-layer donor (D) and recipient (R) cells. S-layer shedding cells (JS1001; donors) were cocultured with S-layer-negative cells (recipients) that possess wild-type (JS1003; R⁺) or deficient (JS1004; R⁻) levels of SLPS. Equivalent numbers of wild-type (NA1000; wt) cells alone, donor cells alone, donors plus wild-type SLPS recipients (D+R⁺), or donors plus deficient SLPS recipients (D+R⁻) were subjected to low-pH extraction. (c) S-layer reattachment using soluble RsaA from the supernatant of shedder cultures. Soluble RsaA harvested from the supernatant of an S-layer-shedding strain (JS1001) was incubated with a corresponding number of S-layer-negative cells that possess wild-type (wt) or deficient (d) levels of SLPS. Reattached RsaA was subsequently extracted by low-pH treatment. Lane 1 shows results of the 12-μl supernatant from the shedder strain. In lanes 2 and 3, to assess the extent of S-layer reattachment, low-pH extractions of 100% (lane 2) and 10% (lane 3) of the number of recipient cells used in the reattachment assay were performed on NA1000 cells. In lanes 4 to 7, results of low-pH extraction of S-layer-negative cells possessing wild-type levels of SLPS (JS1003; control, lane 4), deficient levels of SLPS (JS1004; control, lane 5), wild-type levels of SLPS (JS1003) after incubation with soluble RsaA (lane 6), and deficient levels of SLPS (JS1004) after incubation with soluble RsaA (lane 7) are shown.

Coculturing of an S-layer donor strain (with defective LPS that sheds S-layer protein) and an S-layer recipient strain (that possesses normal LPS but lacks the S-layer protein) achieves S-layer recrystallization in A. salmonicida (18). Accordingly, an S-layer-shedding strain (JS1001, donor) was cocultured with an S-layer-negative strain that possessed wild-type (JS1003, recipient) or deficient (JS1004, recipient) levels of SLPS. More S-layer reassembled selectively to SLPS-positive cells (JS1003) in this method compared to the previous methods (Fig. 1b). These results suggested that soluble, reassembly-competent RsaA was present in the supernatant of S-layer-shedding strains, in addition to the RsaA that forms aggregates. Accordingly, supernatant from an S-layer-shedding C. crescentus culture (JS1001) was analyzed, and soluble RsaA was indeed found in appreciable quantity (Fig. 1c, lane 1). RsaA obtained in this manner reassembled onto S-layer-negative (JS1003) cells at wild-type (NA1000) levels (Fig. 1c, compare lane 6 to lane 2).

Thus, the method of RsaA preparation strongly affected the ability of the protein to reassemble on the cell surface, and it was apparent that soluble secreted RsaA was the best source of RsaA variants to test regions or residues that might be important for RsaA anchoring.

SLPS is required for S-layer anchoring.Strains deficient in SLPS (such as JS1001) shed their S-layer into the culture medium. JS1001 was isolated as a strain able to grow in the absence of calcium which acquired an as yet undefined mutation that greatly reduced, but did not eliminate, SLPS production (2). In reattachment assays of RsaA to JS1004 (S-layer-negative version of JS1001), a low level of RsaA still reattached to the cells (Fig. 1c, lane 7, for example), maybe due to a low level of SLPS still present.

The N-acetylperosamine biosynthetic pathway was previously proposed (2), and disruption of manB, which encodes a putative phosphomannomutase involved in N-acetylperosamine synthesis, results in the loss of SLPS production (2). We disrupted manB in RsaA-negative strain JS1013, creating JS1014, a strain completely devoid of SLPS (Fig. 2a, lane 4). To determine if RsaA reattachment requires SLPS, soluble, reattachment-competent RsaA (isolated from the supernatant of JS1001) was incubated with S-layer-negative cells that had no SLPS (JS1014), were deficient in SLPS (JS1004), or had wild-type levels (JS1003). No RsaA reattachment was observed to JS1014, a small amount of RsaA reattached to JS1004, and a large amount of RsaA reattached to JS1003 (Fig. 2b), indicating that SLPS is required for S-layer anchoring.

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FIG. 2.

SLPS and the attachment of S-layer. (a) SLPS levels in SLPS-positive, -deficient, and -negative C. crescentus strains. Immunoblot analysis using SLPS antiserum. Whole-cell protein preparations from equivalent numbers of cells were used. Lane 1, Km resistance cassette interruption of rsaA in spontaneous calcium-independent mutant (deficient levels of SLPS; JS1004); lane 2, Km resistance cassette interruption of rsaA (wild-type levels of SLPS; JS1003); lane 3, wild-type C. crescentus, NA1000; lane 4, manB knockout in S-layer-negative strain (no SLPS; JS1014). Presence (+) or absence (−) of manB and S-layer (SL) is indicated. (b) S-layer attachment requires SLPS. Immunoblot analysis using RsaA antiserum. Wild-type RsaA was incubated with S-layer-negative cells possessing various SLPS levels: wild-type (JS1003), ++; deficient (JS1004), +; or none (JS1014), −. Whole-cell protein preparations from equivalent numbers of cells were then performed. The lane marked control (ctl) is input RsaA alone. N/A, not applicable.

Region of RsaA that mediates S-layer anchoring.Since disruption of the putative RsaA anchoring or crystallization domains results in S-layer shedding, when a mutation results in a shedding phenotype, it is difficult to know whether RsaA anchoring or crystallization has been perturbed. The goal, then, was to identify a portion of RsaA that was sufficient for anchoring yet small enough that it would likely be able to do so only by direct interaction with the cell surface, rather than by RsaA subunit-subunit interactions.

To that end, rsaA variants with collagenase cleavage sites at selected positions in RsaA were constructed. Soluble protein was isolated from culture supernatants of shedder strains and treated with collagenase, and the resulting fragments were incubated with S-layer-negative cells. Initial results from several experiments suggested that both the RsaA N- and C-terminal fragments reattached to S-layer-negative cells (not shown). Upon closer inspection, it appeared that the RsaA N-terminal fragment always reattached, while the RsaA C-terminal fragment only reattached when residual full-length (uncleaved) RsaA was available and also reattached (not shown). This suggested that the reattachment of the RsaA C-terminal fragment may be due to an association of C-terminal fragments with residual full-length RsaA rather than to direct attachment of the RsaA C-terminal fragment to the cell surface. To test this hypothesis, RsaA bearing a collagenase cleavage site at residue 277 was treated with collagenase for various lengths of time to generate partial and complete digests of the protein. The digested RsaA preparations were then incubated with S-layer-negative cells (JS4015) to assess attachment. As hypothesized, the RsaA C-terminal fragment (residues 278 to 1026) only reattached when some full-length RsaA was available and also reattached (Fig. 3, compare lane 2 with lane 5), suggesting that the RsaA C-terminal fragment contained no anchoring information. In addition, while smaller RsaA N-terminal fragments could not be readily detected with RsaA antiserum (data not shown), these data also support the conclusion that residues 1 to 277 RsaA of [Rsa(1-277)] is sufficient for RsaA anchoring. Note that RsaA(1-277) appears to run anomalously at ∼34 kDa rather than the expected ∼28 kDa. The weak band at ∼28 k is likely due to a low level of Sap activity still present in JS4015; this point is further discussed below.

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FIG. 3.

RsaA C-terminal fragment anchoring requires full-length RsaA. Immunoblot analysis was performed using RsaA antiserum. RsaA bearing a collagenase cleavage site at residue 277 was treated with collagenase for increasing lengths of time. The resulting RsaA fragments were incubated with Sap-negative, S-layer-negative cells (JS4015). After a washing step, preparations from equivalent numbers of cells were examined. Lane 1, JS4015 cells (no treatment, N/T); lanes 2 to 5, JS4015 cells plus RsaA digested by collagenase for various lengths of time (60, 120, or 180 min or overnight); lane 6, RsaA digested by collagenase overnight. O/N, overnight.

Investigation of regions within the RsaA anchoring domain.Initial experiments attempting to reattach full-length RsaA with various N-terminal mutations were unsuccessful (data not shown) and served as preliminary evidence that RsaA anchoring information is located in the RsaA N terminus. To discriminate regions within the RsaA N terminus that are important for anchoring, independent mutations in the N terminus were constructed at positions in RsaA corresponding to amino acids 7, 29, 69, 154, 169, and 222 (Fig. 4a). Mutations at residues 7, 154, and 222 resulted in a two-amino-acid exchange for Gly/Ser, while mutations at residues 29, 69, and 169 resulted in the insertion of four amino acids (N-Asp-Gly-Ser-Val) at these positions. All of these constructs also possessed the collagenase cleavage site at residue 277. Full-length RsaA from the constructs was collected from the C. crescentus shedder strain JS1012 and treated with collagenase. After collagenase treatment, all constructs yielded RsaA fragments of the expected size, as evidenced by immunoblotting (data not shown). The resulting fragments were incubated with Sap-negative (see next section), S-layer-negative cells that were either SLPS positive (JS4015) or negative (JS4024). After washing, the cells were analyzed for the presence of RsaA and RsaA fragments. No RsaA fragments were detected on the SLPS-negative cells, as expected (Fig. 4b). The mutations at RsaA positions 7, 29, and 69 caused loss of anchoring to the SLPS-positive cells (Fig. 4b), as did mutations at positions 154, 169, and 222 (not shown), suggesting that all of the regions of RsaA that were mutated contribute to the anchoring of the RsaA N terminus to SLPS. The digest of RsaA bearing the collagenase site at residue 277, but without further mutation in the N terminus (the control input protein), appeared to go to completion (Fig. 4b, lane ctl). The resulting RsaA(1-277) fragment clearly reattached to SLPS-positive cells, and the C-terminal fragment [RsaA(278-1026)] did not (Fig. 4b, wt lanes). The lack of C-terminal fragment reattachment is consistent with our previous finding that some full-length protein is required for any C-terminal fragment to bind and that only the N terminus of RsaA carries the anchoring information for the protein (Fig. 3). Taken together, these data confirm that the RsaA N terminus mediates RsaA anchoring, and small perturbations within the first ∼225 amino acids disrupt RsaA anchoring.

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FIG. 4.

The effect of mutations in the N terminus of RsaA. (a) Linear representation of mutant RsaA used for reattachment studies. Independent mutations at positions in the RsaA N terminus corresponding to amino acids 7, 29, 69, 154, 169, and 222 were constructed. These full-length proteins were isolated from the C. crescentus shedder strain JS1012 and treated with collagenase. The resulting RsaA fragments were assessed for their ability to reattach to S-layer-negative cells. (b) Mutations at RsaA amino acids 7, 29, and 69 disrupt RsaA attachment. After collagenase digestion, RsaA fragments were incubated with Sap-negative cells that either possessed (JS4015; +) or did not possess (JS4024; −) SLPS. After a washing step, whole-cell protein preparations from equivalent numbers of cells were examined by immunoblot analysis using RsaA antiserum. The lane marked control (ctl) is collagenase-treated input RsaA that did not possess an additional mutation in its N terminus. wt, wild type.

Sap localization and proteolytic activity.In initial RsaA reattachment studies, we observed that reattachment of RsaA(1-277) to the cell surface of S-layer-deficient cells also resulted in a single cleavage of RsaA(1-277), such that a shorter product than predicted was noted in the assay. Interestingly, this product of about 28 kDa was the same size as cleavage products previously observed for some other RsaA mutants cleaved by the S-layer-associated protease, Sap (8); the working hypothesis is that this represents a “weak” site that can be cleaved by Sap if RsaA folding results in incomplete or abnormal secondary structure.

To determine if Sap was responsible for the cleavage of RsaA(1-277), soluble RsaA bearing the collagenase site at residue 277 was isolated from either Sap-positive (JS1004) or Sap-negative cells (JS1012). The isolated protein was treated with collagenase, and the resulting RsaA fragments were incubated with S-layer-negative target cells that were either Sap positive (JS1003) or Sap deficient (JS4015). The source of the input protein did not matter in terms of the cleavage of RsaA(1-277), however; the use of Sap-positive target cells led to complete cleavage of this protein, whereas the use of Sap-deficient target cells largely prevented this cleavage (Fig. 5, compare lanes 1 and 4 or lanes 2 and 5). In JS4015, the Sap deficiency derives from a point mutation in the active site (38), which reduces its activity but may not abolish it completely and so may allow for some residual cleavage of RsaA(1-277). This may account for the presence of a small amount of the cleaved version of RsaA(1-277) when JS4015 cells were used as target cells for RsaA(1-277) reattachment (Fig. 5, lanes 1 and 2). The band that is smaller than the cleaved RsaA(1-277) (lanes 4 and 5) and that does not appear in lanes 1 and 2 may result from a poorly recognized cleavage site that is only exposed after an initial cut of RsaA(1-277) by Sap.

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FIG. 5.

Sap cleaves RsaA(1-277). RsaA bearing a collagenase site at 277 was isolated from a Sap⁻ (JS1012) or Sap⁺ (JS1004) shedder (S-layer secreting) strain and then treated with collagenase. The resulting RsaA fragments were then incubated with corresponding numbers of S-layer-negative target cells that are Sap⁻ (JS4015) or Sap⁺ (JS1003). After a washing step, whole-cell preparations were examined by immunoblot analysis using RsaA antiserum.

These data suggest that Sap harbored by the target cells was responsible for the cleavage of RsaA(1-277). This was a fortuitous result, since the S-layer reattachment assay involved mutating sites within RsaA(1-277) and then testing these fragments for their ability to reattach to S-layer-negative cells. We wanted to be sure that when a particular RsaA mutation led to loss of RsaA anchoring, this loss of anchoring resulted from the fact that the sequence that was mutated contributed directly to anchoring, either by direct attachment of this sequence to SLPS or by contributing to the RsaA structure required for anchoring, rather than simply exposing a previously unrecognized cleavage site. To that end, in the reattachment assays involving mutant RsaA (described above), S-layer-negative strains that are also Sap negative (e.g., JS4015 and JS4024) were used as targets for reattachment (Fig. 4b).

Since the supernatant of the target cell culture was removed prior to exogenous addition of RsaA(1-277) and since RsaA(1-277) is not likely to be imported into the cell, for Sap to be able to cleave RsaA(1-277), it must either be located on the cell surface or released from the cytoplasm in some way. To rule out the possibility that the lysis of some reattachment target cells released Sap from the cytoplasm, allowing it to encounter and cleave RsaA(1-277), some reattachment mixtures were boiled immediately after reattachment of RsaA(1-277), with the expectation that boiling would denature any protease that was hypothetically released from lysed cells. Samples prepared in this way still exhibited the proteolysis of RsaA(1-277) (data not shown), suggesting that the Sap-mediated proteolysis occurred during the reattachment process rather than some time after reattachment due to some cell lysis. These results suggest that Sap is located on the cell surface.

Secretion of Sap to the cell surface.Given the indication that Sap was surface localized, focus shifted to assessing how the protease was secreted. There is no significant degree of homology between the C-terminal secretion signal of RsaA and the C terminus of Sap. However, AprA, a protease from P. aeruginosa whose C terminus shares even less homology to the C terminus of RsaA, can be secreted by the rsaA type I transporter (1); we considered it possible that Sap might also be secreted by this system.

Accordingly, an NA1000-derived strain was constructed that was null for rsaF _a and rsaF _b, two transporter genes required for the C. crescentus S-layer type I secretion system (37) and for rsaA (JS1011) (Fig. 6a, lane 3); a JS4000-derived strain that was null for rsaF _a and rsaF _b (JS4025) (Fig. 6a, lane 6) was also constructed. These strains were used as targets for reattachment of RsaA(1-277) to see if Sap proteolytic activity was affected. Cleavage of RsaA(1-277) was significantly reduced when JS1011 was used compared results with a target (UJ2602) that possesses the wild-type outer membrane proteins (OMPs) of the S-layer type I transporter proteins (Fig. 6b, compare lanes 3 and 4). This suggests that RsaF_a and/or RsaF_b is involved in the secretion of Sap in strain NA1000, and thus when the OMPs are not present, Sap largely does not get secreted and therefore cannot access and cleave RsaA(1-277) that has reattached to the cell surface.

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FIG. 6.

The effect of RsaF_a and RsaF_b levels on Sap secretion. (a) RsaF_a and RsaF_b levels in wild-type and RsaF knockout strains. Whole-cell protein preparations from equivalent numbers of NA1000 (wt) or JS4000 (wt) cells or strains derived from them that are null for RsaF_a (F_a⁻; JS4023), RsaF_b (F_b ⁻; JS1008), or both RsaF_a and RsaF_b (F_a ⁻/F_b ⁻; JS1011 and JS4025) were examined by immunoblot analysis using RsaF antiserum. (b) S-layer type I secretion OMP RsaF_a and/or RsaF_b is involved in the secretion of Sap. RsaA bearing a collagenase cleavage site at residue 277 was isolated from the Sap⁻ C. crescentus shedder strain JS1012 (lane 2) and then treated with collagenase (lane 1). The resulting RsaA fragments were then incubated with corresponding numbers of S-layer-negative C. crescentus cells derived from NA1000 that either possess (UJ2602, lane 4) or do not possess (JS1011, lane 3) RsaF_a and RsaF_b. After washing, cells were immediately boiled in the presence of SDS-PAGE loading buffer. Equal loadings were examined by immunoblot analysis using RsaA antiserum. (c) A third RsaF OMP may be present in strain JS4025. RsaA bearing a collagenase cleavage site at residue 277 was isolated from the Sap⁻ C. crescentus shedder strain JS1012 and then treated with collagenase (lane 4). The resulting RsaA fragments were then incubated with corresponding numbers of cells from JS1011 (lane 1) or JS4025 (lane 2). As a control, the RsaA fragments were also incubated with JS4015 cells (lane 3). After washing, cells were immediately boiled in the presence of loading buffer. Equal loadings were examined by immunoblot analysis using RsaA antiserum.

Strain JS4000 is a strain of distinct lineage from NA1000, though it is capable of producing an RsaA that is identical to that of NA1000, except for an apparent spontaneous frameshift mutation in rsaA that results in an early stop codon (33). A search of the NA1000 genome (www.tigr.org ) suggested that there are only two chromosomal OMP genes (rsaF _a and rsaF _b) involved in S-layer type I secretion, and this was confirmed experimentally (37). In contrast to JS1011 (derived from NA1000), upon introduction of a plasmid-borne copy of rsaA into the JS4000-derived JS4025, RsaA was still secreted (not shown). This suggested that a third rsaF gene may be present on the JS4000 chromosome, allowing for RsaA secretion when rsaF _a and rsaF _b have been inactivated. To gather more evidence of a third RsaF OMP in JS4000 and to demonstrate that Sap can utilize this putative OMP for secretion, cleavage of RsaA(1-277) was assessed using JS4025 as a target for reattachment of RsaA(1-277). Full cleavage of RsaA(1-277) was observed when JS4025 (Fig. 6c, lane 2) but not JS1011 (Fig. 6c, lane 1) was used as a target. Taken together, these data suggest that a third RsaF protein is present in JS4000, and this third RsaF transporter protein is also involved in the secretion of Sap.